While proteins produced in prokaryotes, for example Escherichia coli, have no carbohydrate chain, proteins and lipids produced in eukaryotes such as yeast, fungi, plant cells and animal cells have a carbohydrate chain bound thereto in many instances.
Carbohydrate chains bound to proteins in animal cells include N-glycoside bond type carbohydrate chains (also called N-glycans) bound to an asparagine (Asn) residue in the protein and O-glycoside bond type carbohydrate chains (also called O-glycans) bound to a serine (Ser) or threonine (Thr) residue. It has recently been revealed that a certain kind of lipid containing a carbohydrate chain is covalently bound to a number of proteins and that those proteins are attached to the cell membrane through the lipid. This carbohydrate chain-containing lipid is called glycosyl phosphatidylinositol anchor.
Other carbohydrate chains, including glycosaminoglycans, are also present in animal cells. Compounds comprising a protein covalently bound to a glycosaminoglycan are called proteoglycans. The glycosaminoglycans of the carbohydrate chains of proteoglycans are similar in structure to O-glycans, which are carbohydrate chains of glycoproteins, but differ chemically therefrom. Glycosaminoglycans comprise repeating disaccharide units composed of glucosamine or galactosamine and a uronic acid (except for keratan sulfate which has no uronic acid residue) and have a covalently bound sulfate residue (except for hyaluronic acid which has no sulfate residue).
Further, carbohydrate chains in animal cells are also present in substances called glycolipids. Sphingoglycolipids are one type of glycolipid present in animal cells. Sphingoglycolipids are composed of a carbohydrate, a long-chain fatty acid and sphingosine, a long-chain base, covalently bound together. Glyceroglycolipids are composed of a carbohydrate chain and glycerol covalently bound together.
Recent advances in molecular biology and cellular biology have made it possible to clarify the functions of carbohydrate chains. To date, a variety of functions of carbohydrate chains have been elucidated. First, carbohydrate chains play an important role in the clearance of glycoproteins in blood. It is known that erythropoietin produced by introducing the relevant gene into Escherichia coli retains activity in vitro but undergoes rapid clearance in vivo [Dordal et al.: Endocrinology, 116, 2293 (1985) and Browne et al.: Cold Spring Harbor Symposia on Quantitative Biology, 51, 693 (1986)]. It is known that while native human granulocyte-macrophage colony stimulating factor (hGM-CSF) has two carbohydrate chains of the N-glycoside bond type, a reduction in the number of carbohydrate chains results in a proportional increase in the rate of clearance in rat plasma [Donahue et al.: Cold Spring Harbor Symposia on Quantitative Biology, 51, 685 (1986)]. The rate of clearance and the site of clearance may vary or differ depending on the structure of the carbohydrate chain in question. For example, it is known that hGM-CSF having a sialic acid residue undergoes clearance in the kidney while hGM-CSF deprived of sialic acid shows an in creased rate of clearance and undergoes clearance in the liver. Alpha1-acid glycoproteins differing in carbohydrate structure and biosynthesized in the presence of various N-glycoside type carbohydrate chain biosynthesis inhibitors using a rat liver primary culture system were studied with respect to their rate of clearance from rat plasma and their rate of clearance from rat perfusate. In both cases, the rate of clearance was reduced in the order: high mannose type, carbo hydrate chain-deficient type, hybrid type and composite type (natural type). It is known that the clearance from blood of tissue-type plasminogen activator (t-PA), which is used medicinally as a thrombolytic agent, is greatly influenced by the structure of its carbohydrate chain.
It is known that carbohydrate chains give protease resistance to proteins. For example, when carbo hydrate formation on fibronectin is inhibited with tunicamycin, the rate of degradation of intracellular carbohydrate chain-deficient fibronectin increases. It is also known that addition of a carbohydrate chain may result in increased heat stability or freezing resistance. In the case of erythropoietin and β-interferon, among others, the carbohydrate chain is known to contribute to increased solubility of the protein.
Carbohydrate chains also serve to maintain protein tertiary structure. It is known that when the membrane binding protein of vesicular stomatitis virus is devoid of the two naturally-occurring N-glycoside bond type carbohydrate chains, transport of the protein to the cell surface is inhibited and that when new carbohydrate chains are added to the protein, it is transported. It was revealed that, in that case, intermolecular association of the protein through disulfide bonding is induced following the elimination of carbohydrate chains and, as a result, protein transport is inhibited. When carbohydrate chains are added, association is inhibited, and the proper tertiary protein structure is maintained and protein transport becomes possible. As regards the site of addition of the new carbohydrate, it has been shown that there is a considerable amount of flexibility. In contrast, it has also been shown in certain instances that, depending on the site of carbohydrate chain introduction, the transport of a protein having a natural carbohydrate chain or chains may be completely inhibited.
Examples are also known where a carbohydrate chain serves to mask an antigenic site of a poly peptide. In the case of hGM-CSF, prolactin, inter feron-γ, Rauscher leukemia virus gp70 and influenza hemagglutinin, experiments using a polyclonal antibody or a monoclonal antibody to a specific site on the peptide suggest that carbohydrate chains of these proteins inhibit antibody binding. Cases are also known where carbohydrate chains themselves are directly involved in the expression of activity by aglyco protein. For instance, carbohydrates are thought to be associated with the expression of activity of such glycoprotein hormones as luteinizing hormone, follicle stimulating hormone and chorionic gonadotropin.
Carbohydrate chains serve an important function in the phenomenon of recognition between cells, between proteins or between a cell and a protein. For example, it is known that structurally different carbohydrate chains undergo clearance in vivo at different sites. It has recently been revealed that the ligand of the protein ELAM-1, which is expressed specifically on vascular endothelial cells during an inflammatory response and promotes adhesion to neutrophils, is a carbohydrate chain called sialyl Lewis x [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc; where NeuAc: sialic acid; Gal: galactose; Fuc: fucose; GlcNAc: N-acetylglucosamine]. The possibile use of carbohydrate chains themselves or modifications thereof as drugs or the like is thus suggested [Phillips et al.: Science, 250, 1130 (1990); Goelz et al.: Trends in Glycoscience and Glycotechnology, 4, 14 (1992)]. Like ELAM-1, L-selectin, expressed in some T lymphocytes or neutrophils, and GMP-140 (also called P-selectin), expressed in platelets or on the membrane surface of vascular endothelial cells upon inflammatory stimulation, are associated with inflammatory responses. It is suggested that their ligand may be a carbohydrate chain analogous to sialyl Lewis x, the ELAM-1 ligand [Rosen et al.: Trends in Glycoscience and Glycotechnology, 4, 1 (1992); Larsen et al.: Trends in Glycoscience and Glycotechnology, 4, 25 (1992); Aruffo et al.: Trends in Glycoscience and Glycotechnology, 4, 146 (1992)].
It has been suggested that, in cancer metastatis, as in inflammatory responses, ELAM-1 and GMP-140 cause adhesion of cancer cells to the vascular endothelium or aggregation of cancer cells with platelets and thereby promote cancer metastatis [Goelz et al.: Trends in Glycoscience and Glycotechnology, 4, 14 (1992); Larsen et al.: Trends in Glycoscience and Glycotechnology, 4, 25 (1992)]. This is in agreement with the finding that the level of expression of the sialyl Lewis x carbohydrate chain is high in cancer cells that are highly metastatic [Irimura et al.: Jikken Igaku (Experimental Medicine), 6, 33 (1988)].
Gangliosides constitute a group of cell membrane constituent glycolipids. They are molecules composed of a sialic acid residue-containing carbohydrate chain, which is a hydrophilic side chain, sphingosine, which is a hydrophobic side chain, and a fatty acid. It is known not only that the expression of gangliosides varies with the cell, organ and animal species but also that gangliosides undergo quantitative and qualitative changes during the process of cell differentiation or oncogenesis [Hakomori: Cancer Research, 45, 2405 (1985)]. Scores of gangliosides have been discovered so far, including GM3 which is expressed in a variety of normal cells, and gangliosides occurring in extremely small amounts [Wiegant: Gangliosides and Cancer, Verlagsgesellschaft, 1989, pages 5–15]. GD3, for example, occurs in small amounts in normal tissues but it is expressed at high levels in neuroectodermal tumors, such as malignant melanoma. It is therefore believed to be a type of cancer antigen [Tsuchida et al.: Journal of the National Cancer Institute, 78, 45–54 (1987)]. A recent report shows that the proportions of GD3 and GM3 vary according to the degree of malignancy of malignant melanoma [Ravindranath et al.: Cancer, 67, 3029 (1991)] and it is widely known that GD3 is an important cancer antigen. Furthermore, it has been demonstrated that the expression of GD3 is induced in cells into which an oncogene has been introduced, supporting the close relation between cell transformation and GD3 expression [Sanai et al.: Journal of Biochemistry, 107, 740–742 (1990)]. As for the functions of GD3, it has been suggested that it plays an important role in adhesion of cancer cells to extracellular substrates [Burns et al.: Journal of Cell Biology, 107, 1225–1230 (1988)].
It has been suggested that abnormal expression of GD3 is due to α-2,8-sialyltransferase, which is a GD3 synthetase [Yusuf et al.: Biological Chemistry Hoppe-Seyler, 368, 455–462 (1987)]. Only the partial purification of GD3 synthetase has been reported [Gu et al.: Biochemical and Biophysical Research Communications, 166, 387–393 (1990)]. No GD3 synthetase has been isolated as yet.
Attempts have been made to effect passive immunization of cancer patients by administering a monoclonal antibody to GD3 [Houghton et al.: Proceedings of the National Academy of Sciences of the U.S.A., 82, 1242 (1985)] and to effect active immunization of cancer patients by administering GD3 per se as a vaccine [Portoukalian et al. International Journal of Cancer, 49, 893–899 (1991); Ritter et al.: International Journal of Cancer, 48, 379–385 (1991)]. GD3 is thus a valuable cancer antigen. The quantity of GD3 that can be obtained by purification from tissues, however, is limited [Takamizawa et al.: Journal of Biological Chemistry, 261, 5625–5630 (1986)]. Chemical synthesis of GD3 requires sophisticated techniques and yields are very low [Ito et al.: Journal of the American Chemical Society, 111, 8508–8510 (1989)].
In view of the above-described association of GD3 with oncogenesis or cancer metastasis, it is expected that cancer might be treated by inhibiting the enzymatic activity of the GD3 synthetase α-2,8-sialyltransferase or suppressing expression of the relevant gene. Antisense RNA/antisense DNA techniques [Tokuhisa: Bioscience and Industry, 50, 322 (1992); Murakami; Kagaku (Chemistry), 46, 681 (1991)] and triple helix techniques [Chubb and Hogan: Trends in Biotechnology, 10, 132 (1992) can be used to suppress gene expression specifically. For suppressing expression of a specific glycosyltransferase using the antisense RNA/DNA technique, the gene in question or information about the base sequence of the gene is required. It is thus important to clone the desired glycosyltransferase gene and determine the base sequence of same.
Further, as mentioned above, GD3 synthetase α-2,8-sialyltransferase is associated with oncogenesis and it is thus expected that it could be used in cancer diagnosis, that is, that the level of expression of the synthetase could be used to detect the presence of a tumor. The following can be used to assay expression of the α-2,8-sialyltransferase (GD3 synthetase) gene: Northern hybridization using the gene in a labeled form, for example in a radiolabeled form, as a probe [Sambrook, Fritsch and Maniatis: Molecular Cloning—A laboratory manual, second edition, Cold Spring Harbor Laboratory Press, 1989] and polymerase chain reaction (hereinafter, “PCR”) [Innis et al.: PCR Protocols, Academic Press, 1990]. In applying these techniques, the gene for the GD3 synthetase α-2,8-sialyltransferase or knowledge of the base sequence thereof is required. From this viewpoint as well, it is important to clone the gene for GD3 synthetase α-2,8-sialyltransferase and determine its base sequence.
JP-A-2-227075 discloses the possibility of improving the properties of physiologically active useful proteins, such as granulocyte colony stimulating factor (G-CSF) and prourokinase (pro-UK), by artificially introducing a carbohydrate chain into the proteins using recombinant DNA technology.
It is an important problem from an industrial viewpoint to modify the structure of the carbohydrate chain of a glycoprotein or a glycolipid, or to prepare a specific carbohydrate chain or a modification thereof in large quantities, making use of α-2,8-sialyltransferase activity of the GD3 synthetase.
There have been marked advances in recent years in the means for modifying carbohydrate chain structures. In particular, it is now possible to structurally modify carbohydrate chains using highly specific enzymes (exoglucosidases) that are capable of releasing carbohydrate units one by one from the end of the carbohydrate chain, or glycopeptidases or endoglycosidases that are capable of cleaving the site of binding to the peptide chain without causing any change in either the peptide or carbohydrate chains, and accordingly, to study biological roles of carbohydrate chains in detail. The recent discovery of endoglycoceramidases that are capable of cleaving the glycolipids at the site between the carbohydrate chain and the ceramide [Ito and Yamagata: Journal of Biological Chemistry, 261, 14278 (1986)] has not only made it easy to prepare carbohydrate chains of glycolipids but has also promoted investigations into functions of glycolipids, in particular glycolipids occurring in cell surface layers. Further, it has become possible to add new carbohydrate chains using glycosyl-transferases. Thus, for instance, sialic acid can be added to a carbohydrate chain terminus using sialyltransferase [Sabesan and Paulson: Journal of the American Chemical Society, 108, 2068 (1986)]. It is also possible, using various glycosyltransferases or glycosidase inhibitors, to modify carbohydrate chains that are to be added [Allan et al.: Annual Review of Biochemistry, 56, 497 (1987)]. However, there is no means available for producing glycosyltransferases for use in synthesizing carbohydrate chains. It is desirable to produce glycosyltransferases in large quantities by cloning glycosyl-transferase genes and causing efficient expression of glycosyl-transferases in host cells utilizing recombinant DNA technology.
As far as sialyltransferase is concerned, a gene for an enzyme having β-galactoside α-2,6-sialyl transferase activity has been isolated and the base sequence thereof has been reported [Weinstein et al.: Journal of Biological Chemistry, 262, 17735 (1987)]. As regards an enzyme having β-galactoside α-2,3-sialyltransferase activity, cloning of a gene coding for an enzyme catalyzing the addition of sialic acid to galactose in an O-glycoside bond type carbohydrate chain (carbohydrate chain added to a serine or threonine residue) of glycoproteins has been reported by Gillespie et al. but the base sequence of said gene has not been reported [Gillespie et al.: Glycoconjugate Journal, 7, 469 (1990)]. Weinstein et al. reported a method of purifying an enzyme having β-galactoside α-2,3-sialyltransferase activity from rat liver [Weinstein et al.: Journal of Biological Chemistry, 257, 13835 (1982)]. This method, however, provides the desired enzyme only in very small amounts. This rat liver β-galactoside α-2,3-sialyltransferase gene has been cloned by Wen et al. [Wen et al.: Journal of Biological Chemistry, 267, 21011 (1992)]. There has been no report, however, of the cloning of a gene for human galactoside α-2,8-sialyltransferase. Large scale preparation of a sialyltransferase species having α-2,8-sialyltransferase activity or cloning of a gene for encoding a product having sialyltransferase activity has not been reported as yet. Therefore, no means is currently available for large scale preparation of a sialyl transferase having α-2,8-sialyltransferase activity, in particular human galactoside α-2,8-sialyltransferase. Methods of detecting or suppressing expression of the enzyme have also not been established.
It is an object of the present invention to provide a novel α-2,8-sialyltransferase species that would make possible efficient production of the ganglioside GD3, a cDNA coding for α-2,8-sialyltransferase, and a vector containing that cDNA. Another object is to provide a method of detecting α-2,8-sialyltransferase activity, which method would be useful in the diagnosis or treatment of diseases such as cancer, and a method of suppressing the expression of α-2,8-sialyltransferase.